Preparation and use of (R),(R)-2,2′-bis-methylnaltrexone

ABSTRACT

This invention relates to synthesis of (R),(R)-2,2′-bis-MNTX, as shown in Formula (I), and related methods and products.

RELATED APPLICATIONS

The present application is a continuation of U.S. patent applicationSer. No. 12/865,915, filed Nov. 29, 2010, which is a national stageapplication under 35 U.S.C. §371 of International PCT Application No.PCT/US2008/001660, filed Feb. 6, 2008, the contents of each of which arehereby incorporated by reference in their entirety.

FIELD OF THE INVENTION

The present invention relates to the synthesis of(R),(R)-2,2′-bis-methylnaltrexone and related methods and products.

BACKGROUND OF THE INVENTION

Methylnaltrexone (MNTX) is a quaternary derivative of the opioidantagonist, naltrexone. MNTX exists as a salt, for example, a bromidesalt. The bromide salt of MNTX is also known in the literature as:Methylnaltrexone bromide; N-Methylnaltrexone bromide; Naltrexonemethobromide; Naltrexone methyl bromide; MRZ 2663BR. MNTX was firstreported in the mid-70s by Goldberg et al. as described in U.S. Pat. No.4,176,186. It is believed that addition of the methyl group to the ringnitrogen of naltrexone forms a charged compound with greater polarityand less liposolubility than naltrexone, preventing MNTX from crossingthe blood-brain barrier in humans. As a consequence, MNTX exerts itseffects in the periphery rather than in the central nervous system withthe advantage that it does not counteract the analgesic effects ofopioids on the central nervous system.

MNTX is a chiral molecule, wherein the quaternary nitrogen can possesseither the (R) or (S) configuration. For example, (R)-MNTX refers to amolecule of MNTX having (R) stereochemistry at the quaternary nitrogen,while (S)-MNTX refers to a molecule of MNTX having (S) stereochemistryat the quaternary nitrogen. All of the reported functions of MNTXdescribed in the literature indicate it is a peripheral opioidantagonist. Some of these antagonist functions are described in U.S.Pat. Nos. 4,176,186, 4,719,215, 4,861,781, 5,102,887, 5,972,954,6,274,591, 6,559,158, and 6,608,075, and in U.S. patent applicationpublication numbers 2003/0022909A1, 20040266806, 20040259899 and20050004155. These uses include reducing the side-effects of opioidswithout reducing the analgesic effect of opioids. Such side-effectsinclude nausea, emesis, dysphoria, pruritus, urinary retention, bowelhypomotility, constipation, gastric hypomotility, delayed gastricemptying and immune suppression. The art discloses that MNTX not onlyreduces the side-effects stemming from opioid analgesic treatment butalso reduces the side-effects mediated by endogenous opioids alone (orin conjunction with exogenous opioid treatment) such as gastrointestinaldysfunction including inhibition of gastric emptying, constipation,inhibition of gastrointestinal motility from any cause such as surgery,inflammation or excessive vagal stimulation and other such conditionsincluding, but not limited to, those mentioned above. However, it isunclear from the art whether the MNTX used in these studies was amixture of (R) and (S) stereoisomers or a single stereoisomer.

The art suggests that isolated stereoisomers of a compound sometimes mayhave contrasting physical and functional properties, although it isunpredictable in any particular circumstance. Quaternary narcoticantagonists exhibit such contrasting physical and functional properties,making it important to develop procedures to isolate and identify MNTXas pure (R)-MNTX or (S)-MNTX. Goldberg, et al.'s U.S. Pat. No.4,176,186, and more recently Cantrell, et al.'s WO 2004/043964 A2describe a protocol for the synthesis of MNTX involving thequatemization of a tertiary N-substituted morphinan alkaloid with amethylating agent. However, both Goldberg et al and Cantrell et alremain silent as to the stereoisomer(s) produced by the synthesis. Basedon the method of synthesis described in each, it is unknown whether theMNTX so produced was either (R) or (S) or a mixture of both.Furthermore, (S)—N-methylnaltrexone ((S)-MNTX), in pure form, and amethod of making pure (S)-MNTX had not been described in the literature.Therefore, researchers would have been unable to definitivelycharacterize and distinguish the stereoisomer(s) obtained by theGoldberg et al or Cantrell et al synthesis in the absence of pure(S)-MNTX as a standard.

In addition to the isolation and characterization of each stereoisomerof quaternary narcotic antagonists, it can be desirable to isolate theantagonists from any additional impurities in the manufacture of apharmaceutical composition. Generally, pharmaceutical compositions alsorequire a high level of purity to meet regulated standards for drugquality and purity. For example, in the synthesis of MNTX as describedabove, impurities are often formed, including degradants or by-productsof manufacture, which may hinder the therapeutic effects of MNTX and/ormay be toxic if present in high enough quantity. As such, it isdesirable to have the ability to determine both the stereochemicalconfiguration and the purity of MNTX. To do this, it is important toidentify, isolate, and chemically characterize impurities, which can beused in chromatographic procedures as standards to confirm the purity ofMNTX.

SUMMARY OF THE INVENTION

The present invention relates to the identification, purification, andsynthesis of an impurity of (R)-MNTX. It has been discovered that thiscompound can arise as an impurity either in the manufacturing processfor (R)-MNTX or as a degradant when certain solutions of (R)-MNTX arestored under certain conditions. The compound is(R),(R)-2,2′-bis-methylnaltrexone. This compound is a salt, and willtherefore have counterion(s). This compound can also exist as azwitterion. Accordingly, to one aspect of the invention, there isprovided a compound of Formula (I),

wherein X or Y is a counterion. In some embodiments, each Xindependently can be the same or different counterion or each X can becovalently attached to the other X. In a certain embodiments, Y canindependently can be the same or different counterion. In a certainembodiment a mixture of X and Y can be present. In some embodiments, Xor Y can be halide, sulfate, phosphate, sulfonate, nitrate, carboxylate,or an organic species. In a particular embodiment, X is bromide. In someembodiments, the compound is isolated with at least 0.5% purity, or atleast 1% purity, or at least 5% purity, or at least 10% purity, or atleast 15% purity, or at least 25% purity, or at least 50%, at least 75%purity, at least 95% purity, or, more preferably, at least 97% purity.In some embodiments, the compound is a solid. In other embodiments, thecompound is packaged as a solution in a sealed bottle. In oneembodiment, the compound is packaged as a solution in sealed bottlehaving a septum.

According to another aspect of the invention, there are provided kitsincluding a compound of Formula (I) and indicia in or on the kitindicating that the compound is present in the kit. In some embodiments,the indicia indicates the purity of the compound. In some embodiments,the indicia indicates the chemical structure of the compound byproviding a chemical formula or a structural drawing. In someembodiments, the kit includes a second compound that is(R)-methylnaltrexone, (S)-methylnaltrexone, or another compound,

Another aspect of the present invention provides methods forsynthesizing (R),(R)-2,2′-bis-MNTX comprising reacting a first moleculeof (R)-methylnaltrexone with a second molecule of (R)-methylnaltrexonein the presence of a catalyst and an oxidizing agent in amountssufficient to form a compound of Formula (I). In some embodiments, thecatalyst is present in 25 mol % or less, or, more preferably 10 mol % orless, relative to the amount of the first molecule of(R)-methylnaltrexone. In some embodiments, the oxidizing agent ispresent in a stoichiometric amount relative to the amount of the firstmolecule of (R)-methylnaltrexone. In one embodiment, the catalyst isferric bromide. In one embodiment, the oxidizing agent is hydrogenperoxide. In some embodiments, the method further comprises isolatingthe compound as a solid. In some embodiments, the compound is isolatedwith at least 0.5% purity, or at least 1% purity, or at least 5% purity,or at least 10% purity, or at least 15% purity, or at least 25% purity,or at least 50%, at least 75% purity, at least 95% purity, or, morepreferably, at least 97% purity. In some embodiments, the compound is asolid.

The present invention also provides methods for determining an impurity,comprising injecting a reference solution comprising a compound ofFormula (I) into an HPLC column under a set of conditions to obtain afirst HPLC chromatogram, wherein the amount and/or chemical identity ofthe compound present in the reference solution is known; injecting asample solution comprising methylnaltrexone into the HPLC column undersaid set of conditions to obtain a second HPLC chromatogram; anddetermining the presence and/or the amount of the compound in the samplesolution. In some embodiments, the reference solution is injectedmultiple times. In some embodiments, the determining comprises comparingretention times of peaks in the first HPLC chromatogram and peaks in thesecond HPLC chromatogram to determine the presence of the compound inthe sample solution. In other embodiments, the determining comprisesquantifying peak areas of the sample solution and peak areas of thereference solution on the HPLC chromatograms and estimating from thesethe amount of the compound in the sample solution. The sample solutioncan comprise (R)-methylnaltrexone and/or (S)-methylnaltrexone, or amixture of (R)-methylnaltrexone and (S)-methylnaltrexone. In someembodiments, the HPLC column is a reverse phase column and the column iseluted using a mobile phase comprising water, methanol, trifluoroaceticacid, or mixtures thereof.

The present invention also provides methods for determining an impurityin a material consisting essentially of methylnaltrexone, comprisinginjecting into an HPLC column, in a single or series of injections, asample solution containing the material and spiked with a referencecompound having a known chemical structure of Formula (I); obtaining anHPLC chromatogram; and determining the presence and/or the amount of thecompound in the material. The sample solution can comprise(R)-methylnaltrexone, or a mixture of (R)-methylnaltrexone and(S)-methylnaltrexone. In some embodiments, the HPLC column is a reversephase column and the column is eluted using a mobile phase comprisingwater, methanol, trifluoroacetic acid, or mixtures thereof. The methodmay further comprise documenting in a written form the chemical identityof the compound and the amount of the compound as an impurity.

The present invention also provides methods for determining an impurityin a material consisting essentially of methylnaltrexone, comprisinginjecting, in a single or series of injections, a solution in which thematerial is dissolved into an HPLC column and obtaining an HPLCchromatogram; determining the amount in the material of a compound knownto have the structure of Formula (I); and documenting in a written formthe chemical identity of the compound and the amount of the compound asan impurity in the material. In some cases, the amount in the materialof the compound is determined by (i) identifying a peak on thechromatogram that corresponds to a peak on a control chromatogram of acompound known to have the structure of Formula (I), (ii) identifying apeak on the chromatogram that corresponds to a relative retention timeof a compound known to have the structure of Formula (I), and/or (iii)identifying a peak on the chromatogram that corresponds that correspondsto a known amount of a spike of the compound known to have the structureof Formula (I). The sample solution can comprise (R)-methylnaltrexone,or a mixture of (R)-methylnaltrexone and (S)-methylnaltrexone. In someembodiments, the HPLC column is a reverse phase column and the column iseluted using a mobile phase comprising water, methanol, trifluoroaceticacid, or mixtures thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a synthetic scheme for the synthesis of(R),(R)-2,2′-bis-methylnaltrexone ((R),(R)-2,2′-bis-MNTX), according toone embodiment of the invention.

FIG. 2 shows an alternative synthetic scheme for the synthesis of(R),(R)-2,2′-bis-MNTX, according to one embodiment of the invention.

DETAILED DESCRIPTION

The present invention provides synthetic routes for the synthesis of(R),(R)-2,2′-bis-methylnaltrexone ((R),(R)-2,2′-bis-MNTX), and relatedproducts and methods.

(R),(R)-2,2′-Bis-MNTX has the structure in Formula (I):

wherein X or Y is a counterion. In some cases, each X independently canbe the same or different counterion. Each X can be covalently attachedto the other X. The counterion can be, for example, a halide (e.g.,iodide, bromide, chloride or fluoride), sulfate, phosphate, nitrate, oran anionic-charged organic species (e.g., sulfonates, such as mesylate,besylate, tosylate, triflate, and the like, carboxylates, such asformate, acetate, citrate, fumarate, and the like). In one embodiment,the counterion is a halide, such as bromide.

The present invention provides methods for the synthesis of(R),(R)-2,2′-bis-MNTX. FIG. 1 shows the synthesis of(R),(R)-2,2′-bis-MNTX, according to one embodiment of the invention. Thephenolic ring of a first (R)-MNTX molecule may be coupled with anotherphenolic ring of a second (R)-MNTX molecule in the presence of acatalyst and an oxidizing agent in amounts sufficient to produce(R),(R)-2,2′-bis-MNTX. Alternatively, the phenolic ring of a naltrexonemolecule may be coupled with another phenolic ring of a secondnaltrexone molecule under similar coupling conditions, and the resultingnaltrexone dimer may be alkylated at the N-substituted tertiary amineswith a methylating agent to form (R),(R)-2,2′-bis-MNTX, as shown in FIG.2.

Because 2,2′-bis-MNTX contains two chiral quaternary amines, it maypossess a number of different stereochemical configurations (e.g.,(R),(R)-2,2′-bis-MNTX, (S),(S)-2,2′-bis-MNTX, or (R),(S)-2,2′-bis-MNTX)based on the stereochemistry of the individual MNTX molecules which arecoupled to form the 2,2′-bis-MNTX dimer. Methods described hereinutilize MNTX with at least 99.5% in the (R) configuration as thestarting material, such that the (R),(R)-2,2′-bis-MNTX isomer is themajor isomer formed. While other isomers of 2,2′-bis-MNTX may possiblybe formed, they would be present in very low or negligible quantities.The synthesis of pure (R)-MNTX is described in U.S. patent applicationSer. No. 11/441,395, filed May 25, 2006, Publication Number US2007-0099946 A1. The stereochemical configuration of (R)-MNTX may bedetermined by using pure (S)-MNTX as a reference standard. The synthesisof pure (S)-MNTX is described in U.S. patent application Ser. No.11/441,452, filed May 25, 2006, Publication Number US 2007-0265293 A1.(R)-MNTX has been found to be an opioid antagonist, while (S)-MNTX is apartial opioid agonist.

In some cases, methods of the invention comprise a coupling reaction,such as the oxidative coupling of aryl groups (e.g., phenols). Thecoupling reaction can involve use of a catalyst (e.g., ferric bromide)that is capable of oxidatively catalyzing the formation of a covalentbond between two molecules, such as two aryl molecules. The catalyst maycomprise a metal, such as iron. Examples of catalysts include, but arenot limited to, iron salts including potassium ferrocyanide, ferricchloride, and ferric bromide, or other coupling agents known to those ofordinary skill in the art. In some embodiments, it is preferred that thecatalyst be present in the reaction in a sub-stoichiometric amount(e.g., catalytic amount) relative to the substrate (e.g., MNTX,naltrexone). For example, the catalyst may be present in 25 mol % orless, more preferably 10 mol % or less, relative to the amount of MNTXor naltrexone. In one embodiment, the catalyst is ferric bromide and ispresent in the reaction in 10 mol %. Catalysts are well known to thoseof ordinary skill in the art and are described extensively in both thepatent literature and in chemistry text books. Appropriate catalysts canbe selected with no more than routine skill by one of ordinary skill inthe art.

An oxidizing agent may also be included in the coupling reaction. In oneembodiment, the oxidizing agent is hydrogen peroxide. Without wishing tobe bound by theory, one possible mechanism for the coupling reaction maycomprise regeneration of the catalytic species by the oxidizing agentupon formation of the dimer molecule (e.g., (R),(R)-2,2′-bis-MNTX). Forexample, the catalyst may be reduced to a catalytically inactivemolecule upon formation of the dimer molecule. The oxidizing agent maythen be used to, for example, oxidize the catalytically inactivemolecule to regenerate the active form of the catalyst. In someembodiments, the oxidizing agent may be hydrogen peroxide. It may bepreferred that the oxidizing agent be present in the reaction in astoichiometric amount (e.g., one mole equivalent) relative to thesubstrate (e.g., MNTX, naltrexone). Optionally, oxygen may be bubbledthrough the reaction mixture to promote the coupling reaction.

The coupling reaction may be performed in any organic solvent, mixtureof organic solvents, or mixture of organic solvent(s) and water. In someembodiments, the reaction may be performed in acidic conditions. In someembodiments, the reaction may be performed in basic conditions. In oneembodiment, the coupling reaction is performed in a mixture of water,methanol, and trifluoroacetic acid. Also, the coupling reaction may beperformed at any temperature between 0° C. and room temperature. In somecases, the second agent may be added to the reaction mixture at 0° C.over a period of time, and the resulting mixture is warmed to roomtemperature. The coupling reaction may proceed from 1 hour to about 10hours, preferably about 1 hour to about 5 hours, more preferably about 1to 2.5 hours, most preferably about 1.5 hours. The reaction can bemonitored using high performance liquid chromatography (HPLC).

In some cases, methods of the invention may further comprise amethylation reaction, such as methylation of the N-substituted tertiaryamines of a naltrexone dimer (FIG. 2). The methylation reaction caninvolve use of a methylating agent, such as methyl bromide. As usedherein, the term “methylating agent” refers to a reactive species havingelectrophilic properties that is capable of introducing a methyl groupat the nitrogen atom of naltrexone, so as to form a covalent bondtherewith. Illustrative methylating agents can be represented by theformula CH₃Z, wherein “Z” is a leaving group which, upon its departure,enables a covalent bond to be formed between the methyl group and thenitrogen atom of naltrexone, forming MNTX. Methylating agents andleaving groups are well known to those of ordinary skill in the art andare described extensively in both the patent literature and in chemistrytext books. Suitable Z groups include, but are not limited to, fluoro,chloro, bromo, iodo, —OSO₂CF₃, CH₃OSO₂O—, —OSO₂CH₃, —OSO₂C₆H₄-p-CH₃,—OSO₂C₆H₄-p-Br.

The method can further involve purification of (R),(R)-2,2′-bis-MNTXusing at least one purification technique, such as chromatography orcrystallization. The chromatography can be reverse-phase chromatography,regular-phase chromatography, and/or ion exchange chromatography. Insome embodiments, the regular-phase chromatography may involve use of analumina or silica gel column. In some cases, methods of the inventionmay involve use of a reverse phase HPLC column followed by ion exchangechromatography. For reverse phase chromatography, the HPLC column may beeluted using a mobile phase comprising water, methanol, trifluoroaceticacid, or mixtures thereof. The crystallization solvent can be an organicsolvent, a mixture of organic solvents, or a mixture of organicsolvent(s) plus water. A preferred solvent can be an alcohol, such asmethanol. Methods for both chromatography and crystallization are knownto those of ordinary skill in the art.

In some cases, the HPLC column is a reverse phase column and the columnis eluted using a mobile phase comprising water, methanol,trifluoroacetic acid, or mixtures thereof.

In an illustrative embodiment, the coupling reaction was carried outwith a catalytic amount of ferric bromide and a stoichiometric amount ofhydrogen peroxide. The procedure was carried out with 8.5 g of (R)-MNTXto produce 660 mg of >97% pure product in 7.8% yield after reverse phasechromatographic separation, followed by ion exchange chromatography andcrystallization from methanol, as described more fully below. It shouldbe understood that one of ordinary skill in the art can optimize themethodologies described herein to obtain higher purity and/or higheryield of (R),(R)-2,2′-bis-MNTX, in which the crystallized product isgreater than 99%, or even greater than 99.9%, pure. The relativeretention times for (R),(R)-2,2′-bis-MNTX, (R)-MNTX and (S)-MNTX are1.55, 1.00, and 0.89, respectively, as determined by HPLC Method B,described herein.

Where purity of a solid is concerned, purity is the weight percent ofthe (R),(R)-2,2′-bis-MNTX versus that of other compounds in the solid.Where purity of a solution is concerned, purity is the weight percent ofthe (R),(R)-2,2′-bis-MNTX versus that of other compounds in thesolution. Purity may be determined by HPLC, wherein the percent purityof a compound refers to the weight percent of the compound present insolution relative to the weight percent of other compounds present inthe same solution.

The present invention may advantageously provide (R),(R)-2,2′-bis-MNTXin sufficient purity in order to enable its use as a reference orstandard in various analytical methods (e.g., HPLC), as described morefully below. In some embodiments, (R),(R)-2,2′-bis-MNTX may be isolatedwith at least 0.5% purity, at least 1% purity, at least 5% purity, atleast 10% purity, at least 15% purity, at least 25% purity, at least 50%purity, at least 75% purity, at least 95% purity, more preferably, withat least 97% purity. In some embodiments, (R),(R)-2,2′-bis-MNTX may beisolated and/or packaged as a solid. In some embodiments,(R),(R)-2,2′-bis-MNTX can be packaged as a solution in a container suchas a sealed bottle or vial, or a sealed bottle comprising a septum.

In another aspect, the present invention provides methods fordetermining the presence and/or amount of (R),(R)-2,2′-bis-MNTX in asample (e.g., a sample comprising (R)-MNTX). For example,(R),(R)-2,2′-bis-MNTX may be formed as an impurity during the synthesisof (R)-MNTX. As used herein, the term “impurity” may refer to degradantswhich arise during storage of (R)-MNTX and/or by-products formed in achemical reaction for manufacturing of (R)-MNTX. In one embodiment, themethod comprises injecting a reference solution comprising(R),(R)-2,2′-bis-MNTX into an HPLC column under a set of conditions toobtain a first HPLC chromatogram wherein the amount and/or chemicalidentity of (R),(R)-2,2′-bis-MNTX present in the reference solution isknown, injecting a sample solution comprising MNTX into the HPLC columnunder the same set of conditions to obtain a second HPLC chromatogram,and comparing the first HPLC chromatogram with the second HPLCchromatogram to determine the presence and/or amount of the impurity.The reference solution may be formed by dissolving a sample (e.g., solidsample) of (R),(R)-2,2′-bis-MNTX in a first solvent, and the samplesolution may be formed by dissolving a solid sample in a second solvent.In some embodiments, the reference solution may contain an additionalcompound(s), wherein the amount and/or identity of the additionalcompound(s) is also known. In one embodiment, the sample (e.g., samplesolution) may comprise (R)-MNTX. It should be understood that theinvention may encompass other samples suspected of containing(R),(R)-2,2′-bis-MNTX.

In one embodiment, the presence of (R),(R)-2,2′-bis-MNTX in the samplesolution may be determined by comparing retention times of peaks in thefirst HPLC chromatogram with the retention times of peaks in the secondHPLC chromatogram. For example, the standard solution comprising(R),(R)-2,2′-bis-MNTX may produce a chromatogram with a peakcorresponding to (R),(R)-2,2′-bis-MNTX and having a particular retentiontime. A sample solution may then be injected into the HPLC column underthe same conditions as the standard solution, and the resultingchromatogram may be studied to determine if a peak exists at the sameretention time as the peak corresponding to (R),(R)-2,2′-bis-MNTX in theHPLC chromatogram of the standard solution. The existence of such a peakcan indicate that (R),(R)-2,2′-bis-MNTX is present in the sample. Inanother embodiment, the amount of (R),(R)-2,2′-bis-MNTX in the samplesolution may be determined by comparing the area of peaks in the firstHPLC chromatogram with the area of peaks in the second HPLCchromatogram, and calculating from these the content of(R),(R)-2,2′-bis-MNTX in the sample solution.

In some embodiments, the present invention provides methods fordetermining an impurity in a material consisting essentially ofmethylnaltrexone, where a sample solution containing the material andspiked with a reference compound having a known chemical structure ofFormula (I), as described herein, is injected into an HPLC column and anHPLC chromatogram is obtained to determine the presence and/or theamount of the compound in the material.

Methods of the invention may further comprise documenting in a writtenform the chemical identity of the compound and the amount of thecompound as an impurity in the material.

In other embodiments, the present invention provides methods fordetermining an impurity in a material consisting essentially ofmethylnaltrexone, wherein a solution in which the material is dissolvedis injected into an HPLC column and an HPLC chromatogram is obtained todetermine the amount in the material of a compound known to have thestructure of Formula (I), as described herein. The chemical identity ofthe compound and the amount of the compound as an impurity in thematerial may then be documented, in a written form. The amount in thematerial of the compound may be determined by (i) identifying a peak onthe chromatogram that corresponds to a peak on a control chromatogram,(ii) identifying a peak on the chromatogram that corresponds to arelative retention time of a compound known to have the structure ofFormula (I), and/or (iii) identifying a peak on the chromatogram thatcorresponds that corresponds to a known amount of a spike of thecompound known to have the structure of Formula (I).

Some embodiments of the invention may be useful in determining theamount and/or presence of (R),(R)-2,2′-bis-MNTX in a sample comprising(R)-MNTX. The sample may be a sample of freshly manufactured material orthe sample may be one stored for a defined period of time. In oneembodiment, a sample of (R)-MNTX may be stored and periodically analyzedusing methods described herein to determine the presence and/or amountof (R),(R)-2,2′-bis-MNTX in the sample which may have been formed by,for example, degradation of (R)-MNTX. In some cases, the sample may beplaced in conditions to intentionally promote degradation of (R)-MNTX,wherein the sample is periodically analyzed using methods describedherein to determine the presence and/or amount of (R),(R)-2,2′-bis-MNTXin the sample. The sample solution may also comprise (R)-MNTX, (S)-MNTX,or mixtures thereof. In one embodiment, the sample solution comprises(R)-MNTX and/or (S)-MNTX. In another embodiment, the sample solutioncomprises a mixture of (R)-MNTX and (S)-MNTX.

In another aspect of the invention, kits are provided containing(R),(R)-2,2′-bis-MNTX. For example, a kit including(R),(R)-2,2′-bis-MNTX may be provided for the purpose of producingstandards for analytical methods, such as HPLC. In one embodiment, thekit may comprise (R),(R)-2,2′-bis-MNTX and indicia in or on the kitindicating that (R),(R)-2,2′-bis-MNTX is present in the kit. In somecases, the indicia indicates the purity of (R),(R)-2,2′-bis-MNTX. Theindicia may also indicate the chemical structure of the compound byproviding a chemical formula or a structural drawing. In some cases, thekit can include a second compound that is (R)-MNTX, (S)-MNTX. or anothercompound The indicia may indicate that the second compound is present inthe kit, or may indicate the purity of the second compound or thechemical structure of the second compound by providing a chemicalformula or a structural drawing. It should be understood that the kitmay also include additional compounds, similarly identified by theindicia.

In some cases, the kit comprises a solution of (R),(R)-2,2′-bis-MNTX. Insome cases, the kit comprises a solid sample of (R),(R)-2,2′-bis-MNTX. A“kit,” as used herein, typically defines a package or an assemblyincluding one or more of the compositions of the invention, and/or othermaterials associated with the invention, such as solvents. Each of thecompositions of the kit may be provided in liquid form (e.g., insolution), or in solid form. In certain cases, some of the compositionsmay be constitutable or otherwise processable, for example, by theaddition of a suitable solvent or other species, which may or may not beprovided with the kit. Examples of other compositions or componentsassociated with the invention include, but are not limited to, solvents,surfactants, diluents, salts, buffers, emulsifiers, chelating agents,antioxidants, drying agents, needles, syringes, packaging materials,tubes, bottles, flasks, beakers, dishes, frits, filters, rings, clamps,wraps, patches, containers, and the like, for example, for using,modifying, assembling, storing, packaging, preparing, mixing, diluting,and/or preserving the compositions components for a particular use, forexample, to a sample.

A kit of the invention may, in some cases, include instructions in anyform that are provided in connection with the compositions of theinvention in such a manner that one of ordinary skill in the art wouldrecognize that the instructions are to be associated with thecompositions of the invention. For instance, the instructions mayinclude instructions for the use, modification, mixing, diluting,preserving, assembly, storage, packaging, and/or preparation of thecompositions and/or other compositions associated with the kit. In somecases, the instructions may also include instructions for the deliveryof the compositions, for example, for a particular use, e.g., to asample. The instructions may be provided in any form recognizable by oneof ordinary skill in the art as a suitable vehicle for containing suchinstructions, for example, written or published, verbal, audible (e.g.,telephonic), digital, optical, visual (e.g., videotape, DVD, etc.) orelectronic communications (including Internet or web-basedcommunications), provided in any manner.

As used herein, “instructions” can define a component of instructionalutility (e.g., directions, guides, warnings, labels, notes, FAQs or“frequently asked questions,” etc.), and typically involve writteninstructions on or associated with the invention and/or with thepackaging of the compounds of the invention. Instructions can alsoinclude instructional communications in any form (e.g., oral,electronic, audible, digital, optical, visual, etc.), provided in anymanner such that a user will clearly recognize that the instructions areto be associated with compounds of the invention.

EXAMPLES Example 1 Synthesis of (R),(R)-2,2′-bis-MNTX

A solution of (R)-MNTX (8.5 g, 0.0195 mol) in 40 mL ofwater:methanol:trifluoroacetic acid (94.9:5:0.1) and ferric bromide(0.57 g, 1.95 mmol) was cooled in an ice/water bath. A solution ofhydrogen peroxide (0.66 g, 0.0195 mol) in 10 mL of water was added tothe solution over a period of 30 minutes. After stirring for 30 minutesin the ice bath, the solution contained 74% (R)-MNTX and 16%(R),(R)-2,2′-bis-MNTX by HPLC analysis. The solution was removed fromthe ice bath and was warmed to room temperature. After 30 min, HPLCanalysis indicated that the solution contained 44% (R)-MNTX, 27%(R),(R)-2,2′-bis-MNTX, and 2% of an impurity at longer retention time.After one additional hour, HPLC analysis indicated that the solutioncontained 27% (R)-MNTX, 35% (R),(R)-2,2′-bis-MNTX, and 6.2% of theimpurity, plus additional impurities.

Example 2 Purification of (R),(R)-2,2′-bis-MNTX

The reaction mixture was loaded directly onto a preparative reversephase column (Biotage 40M C18) and eluted with 2.4 L of a linear solventgradient of water:methanol, with a constant concentration of TFA (95:5water:methanol to 82.5:17.5 water:methanol, in 12 mL fractions). Thefractions were analyzed by HPLC and fractions of similar compositionwere combined in two lots according to purity. The solvents were removedby rotary evaporation under aspirator pressure and then under highvacuum while maintaining a bath temperature below 34° C.

Both lots were subsequently purified separately by reverse phasechromatography on the Biotage 40M C18 column with 2.4 L of a lineargradient of 95:5:0.1 to 70:30:0.1 water:methanol:TFA. The fractions wereanalyzed by HPLC and combined in two lots according to purity. Thesolvents were removed from the impure fractions by rotary evaporationunder aspirator pressure and then under high vacuum while maintaining abath temperature below 34° C. and resubmitted to chromatography asdescribed above, for a total of four iterations of chromatography.

The pure fractions were combined and the solvent was removed asdescribed above to give 1.0 g of (R),(R)-2,2′-bis-MNTX as a yellowglass. A Bio-Rad AG-1-X8 ion exchange column (60 g) was prepared byeluting with 500 mL of 1M HBr and then 4 L of distilled water until pHof the eluant was approximately 6. The product was dissolved in 15 mL ofwater:methanol (95:5) and was loaded onto the freshly prepared ionexchange column. The product was purified using distilled water as theeluant. The product was eluted in the first 100 mL of distilled water.The fractions were combined and the solvent was removed by rotaryevaporation under aspirator pressure with the bath temperaturemaintained at 34° C. The resulting residue was dried on high vacuum lineand was crystallized from 10 mL of methanol to give yellow crystals in7.8% yield (660 mg, >97% pure) after drying under high vacuum.

The product was observed to be unstable in neutral aqueous solution.When dissolved in d⁴-methanol/D₂O, the product transformed to a compoundwith a slightly shorter retention time. However, when stored overnightin water:methanol:TFA (94.9:5:0.1), the product remained stable.

HPLC analysis was performed on a Hewlett Packard 1100 series using HPLCMethod A, described herein.

HPLC Method A:

Column: Phenomonex Intersil ODS-3 column (C18, 5μ, 150×4.6 mm)

Flow rate: 1.5 mL/min

Column temperature: 50° C.

Detector: Diode array detector monitoring @ 280 nm

Elution: Linear gradient

Time (min) % A % B % C Curve 0 0 90 10 initial 45 30 60 10 linear 45.1 090 10 linear 50 0 90 10 hold A = Methanol B = Water C =Water:methanol:TFA (49.5:49.5:1)HPLC Method B:

HPLC Method B is described as “HPLC Method II” in US Publication NumberUS 2007-0099946 A1 (U.S. patent application Ser. No. 11/441,395).

What is claimed:
 1. A method of synthesizing the compound of Formula(I),

comprising: reacting a first molecule of naltrexone with a secondmolecule of naltrexone in the presence of a catalyst in an amountsufficient to form a naltrexone dimer

and reacting the naltrexone dimer with a methylating agent to form acompound of Formula (I), wherein X or Y is a counterion.
 2. The methodof claim 1, wherein each X independently can be the same or differentcounterion or each X can be covalently attached to the other X.
 3. Themethod of claim 1, wherein X or Y is a halide, sulfate, nitrate, or anorganic species.
 4. The method of claim 1, wherein X is carboxylate orsulfonate.
 5. The method of claim 3, wherein the halide is bromide. 6.The method of claim 1, wherein the catalyst is present in 25 mol % orless relative to the amount of naltrexone.
 7. The method of claim 1,wherein the catalyst is present in 10 mol % or less relative to theamount of naltrexone.
 8. The method of claim 1, wherein the catalyst ispresent in a sub-stoichiometric amount relative to the amount ofnaltrexone.
 9. The method of claim 1, wherein the catalyst is selectedfrom the group consisting of a metal, iron, iron salts, potassiumferrocyanide, ferric chloride, ferric bromide, and potassiumhexacyanoferrate (III).
 10. The method of claim 1, wherein the catalystis potassium hexacyanoferrate (III).
 11. The method of claim 1, furthercomprising reacting the first molecule of naltrexone with the secondmolecule of naltrexone in the presence of an oxidizing agent.
 12. Themethod of claim 11, wherein the oxidizing agent is present in astoichiometric amount relative to the amount of naltrexone.
 13. Themethod of claim 11, wherein the oxidizing agent is hydrogen peroxide oroxygen.
 14. The method of claim 11, wherein the oxidizing agent ishydrogen peroxide.
 15. The method of claim 1, wherein the methylatingagent is a compound of the formula CH₃Z wherein Z is a leaving groupthat enables a covalent bond to be formed between the methyl group andthe nitrogen atom of naltrexone.
 16. The method of claim 15, wherein Zis selected from the group consisting of fluoro, chloro, bromo, iodo,—OSO₂CF₃, CH₃OSO₂O—, —OSO₂CH₃, —OSO₂C₆H₄-p-CH₃ and —OSO₂C₆H₄-p-Br. 17.The method of claim 1, wherein the methylating agent is methyl bromide.18. The method of claim 1, further comprising isolating the compound ofFormula (I) as a solid.
 19. The method of claim 1, further comprisingpurifying the compound of Formula (I) by chromatography or bycrystallization.
 20. The method of claim 19, wherein the compound ofFormula (I) is purified by reverse-phase chromatography, regular-phasechromatography and/or ion-exchange chromatography.
 21. The method ofclaim 20, wherein the compound of Formula (I) is purified to at least0.5% purity.
 22. The method of claim 20, wherein the compound of Formula(I) is purified to at least 1% purity.
 23. The method of claim 20,wherein the compound of Formula (I) is purified to at least 5% purity.24. The method of claim 20, wherein the compound of Formula (I) ispurified to at least 10% purity.
 25. The method of claim 20, wherein thecompound of Formula (I) is purified to at least 15% purity.
 26. Themethod of claim 20, wherein the compound of Formula (I) is purified toat least 25% purity.
 27. The method of claim 20, wherein the compound ofFormula (I) is purified to at least 50% purity.
 28. The method of claim20, wherein the compound of Formula (I) is purified to at least 75%purity.
 29. The method of claim 20, wherein the compound of Formula (I)is purified to at least 95% purity.
 30. The method of claim 20, whereinthe compound of Formula (I) is purified to at least 97% purity.